Intestinal ischemia/reperfusion (I/R) results in an intense inflammatory response and microvascular dysfunction, both within the post-ischemic intestine and in distant organs, including the lung and liver. A characteristic feature of this injury response is increased microvascular permeability that ultimately leads to interstitial edema in both the intestine and lungs. Recent studies indicate that products of endothelial cell (e.g., oxidants) and leukocyte activation (proteases and oxidants) are important mediators of reperfusion-induced endothelial barrier dysfunction. It is now recognized that the principal molecular target of these permeability-mediating factors lie within the inter-endothelial tight and adherens junctions. It has been proposed that I/R leads to disorganization of endothelial junctions through several possible mechanisms, which allows for an enhanced permeation of fluid and proteins across the microvasculature. However, how these agents disrupt junctions and barrier are still not well understood. While leukocyte proteases are known to play a role in microvascular injury, it is now clear that tissue derived proteases especially the matrix metalloproteinases (MMPs) may play an equal role in I/R injury. Importantly, it has been recently reported that activated MMPs will degrade endothelial junctions to increase permeability. The work outlined in this project will attempt to determine how MMP synthesis and activation are regulated in post-ischemic endothelial cells and how MMPs contribute to the loss of endothelial barrier function associated with reperfusion. We will use western blotting, RT-PCR, gel zymography and ELISA to measure changes in specific MMP latent content and MMP activity following incubation of endothelial cultures in an hypoxia chamber. We will also use these same techniques to investigate the stability of endothelial junctions under these conditions to test out central hypothesis that MMP mediated disintegration of junctional complexes contributes to barrier dysfunction during reoxygenation. We will study barrier function in vitro using Boyden and Ussing chambers to measure solute and trans-endothelial electrical resistance (TER) following hypoxia/reoxygenation and in the presence of specific MMP inhibitors. Three specific aims are proposed: 1) to define the role of tyrosine kinases and NFkB/PARP in the induction of endothelial MMP synthesis elicited by hypoxia/reoxygenation (H/R), 2) to determine how H/R-induced MMP activation alters endothelial junction organization and 3) to define the contribution of matrix metalloproteinases (MMPs) to the diminished endothelial barrier function observed in post-hypoxic monolayers. We will employ a variety of blockers and inhibitors in this study which will allow us to interfere with either the synthesis and/or activity of MMPs in endothelial cells and evaluate contributions of several MMP related pathways. The proposed studies should significantly advance our understanding of the mechanisms responsible for diminished endothelial barrier function during reperfusion in the intestine and in remote organs as well.